1. Field of the Invention
Systems for the stable transformation of insect cells have been pursued because of their potential to advance the fields of insect genetics, biochemistry, developmental biology, agriculture and medicine. Insect cell culture provides ideal conditions for the study and manipulation of insect genomes in vitro. Increased efforts have been made to isolate transposable and viral elements as vehicles for genetic transformation of insect cells and for use in biotechnology for gene expression in vitro. Thus far, routine gene transfer in insects has been possible only in members of the genus Drosophila (Handler and O'Brochta. 1991. Ann. Rev. Entomol. vol. 36, pp. 159-183), although methods have been sought for generalized transformation of other insect genomes. This invention relates to a segment of a polydnavirus genome which is capable of stable integration into the genome of an insect cell.
2. Description of the Related Art
Cell lines established from a number of insect species, predominantly from the orders Lepidoptera and Diptera [Lynn et al. 1988. In: Invertebrate and fish tissue culture. (Kuroda et al., eds) Japan Scientific Societies Press, Tokyo, pp. 239-242] facilitated the study of insect cellular physiology, endocrinology and biochemistry. Some cell lines have been utilized for studies on insect viruses and have shown varying susceptibilities to infection (Lynn et al., supra). In addition, the manipulation of insect viral replication in insect cells has proven useful. For example, the well-known baculovirus expression vector system (BEVS) has been used extensively for foreign gene expression in insect cells in vitro (King and Possee. 1992. The baculovirus expression system. Chapman & Hall, London), and other insect viruses have been investigated for potential utility as vectors for the introduction of genetic material into insect cell genomes in vitro (Giraud et al., 1992. Virology. vol. 186, pp. 207-218; McKelvey et al. 1996. Biochem. and Biophys. Res. Comm. vol. 225, pp. 764-770; Stoltz et al. 1986. Virology. vol. 155, pp. 120-131, all herein incorporated by reference).
Stable transformation of insect tissues, however, either in vivo or in vitro, has rarely been achieved (Ashburner, M. 1995. Science. vol. 270, pp. 1941-1942), especially the introduction of DNA into the germ line (Ashburner, supra). This event has been achieved thus far only in Drosophila (Rubin and Spradling. 1982. Science. vol. 218, pp. 348-353), by introduction of the P-element, and other transposable elements have recently been successfully used for transformation of the medfly, Ceratitis capitata (Loukeris et al. 1995. Science. vol. 270, pp. 2002-2003; Zweibel et al. 1995. Science. vol. 270, pp. 2005-2008). Because such transformation events require the injection of embryos, methods using viral vectors may be more useful.
Currently, the baculovirus expression system is the most commonly used system for the expression of foreign genes. Although this system is useful for both research and commercial purposes, it has a significant shortcoming, i.e. it can only express foreign genes transiently due to cell death from virus infection.
There is thus a need for a mechanism for the stable transformation of insect cells capable of the continuous expression of a desired protein.